Experimental evaluation of anti inflammatory agents

Dr. Aditi M. Panditrao 1


Experimental Evaluation of Anti Inflammatory Agents


Introduction

• Inflammation – self-defense mechanism

• Protective response intended to eliminate the initial cause of cell injury as well as the necrotic cells and tissues resulting from the original insult

• Symptom of a wide variety of diseases



Phases of Inflammation

• Acute - vasodilatation & increased capillary permeability

• Delayed - infiltration of leukocytes and phagocytes

• Chronic Proliferative - tissue degeneration and fibrosis



Steps in inflammatory response

1. Recognition of the injurious agent2. Recruitment of leukocytes3. Removal of the agent4. Regulation of the response5. Resolution




Anti-inflammatory Analgesic Anti-pyretic

In vitro In vivo

• 3H-Bradykinin receptor binding • 3H-Substance P receptor binding • Assay of PMNLchemotaxis in vitro• PMNL aggregation • Constitutive and inducible cellular arachidonic acid metabolism in vitro• Formation of leukotriene B4 in human white blood cells in vitro• Formation of lipoxygenase products from 14C-arachidonic acid in human PMN in vitro• Formation of eicosanoids from 14C-arachidonic acid in human platelets in vitro

• Methods for testing acute and subacute inflammation• Methods for testing the proliferative phase (granuloma formation)• Side effects of anti-inflammatory agents


In Vitro Methods


3H-Bradykinin receptor binding

• Bradykinin – inflammatory mediator• Bind to B1 and B2 receptor

• B1 receptor – Mast cells, Neutrophils, Macrophages• B2 receptor – Smooth muscle, Sensory nerves,

cardiac tissue


Hageman factor (XII)

Activation

Prekallikrein

HMW Kininogen

LMW Kininogen

Kallikrein

Kallikrein

Bradykinin

Kallidin

Des Arg Bradykinin

Trauma, Tissue injury

B2 • Vasodilation • ↑ Vascular Permeability• Bronchoconstriction• Pain

B1

Kini

nase

I

• Pain •Histamine release• Recruit Neutrophils• IL-1, TNF-α, etc.

INFLAMMATION

Role of Bradykinin in Inflammation


• Tissue homogenates in incubation buffer• Incubated with

• 3H-Bradykinin alone – Total Binding• 3H-Bradykinin + unlabelled Bradykinin (saturated) – Non-specific

Binding• 3H-Bradykinin + Test drug (subtract the non-specific binding)

• Two types – • Competition studies – increasing concentrations of Test drug• Saturation studies – increasing concentrations of 3H-Bradykinin

• Tissues – G. pig ileum, rabbit carotid artery, canine cultured tracheal smooth muscle cells, bovine aortic endothelial cells, human fibroblasts, etc.


3H-Substance P receptor binding

• Substance P – inflammatory mediator• Bind to NK1 and NK2 receptor • Direct and Indirect Actions• ↑ Levels – Acute and Chronic Inflammatory

processes


Trauma, Tissue injury

Peripheral Nerves

Substance P

Direct Actions• Pain • Vasodilation • ↑ Vascular Permeability• Neutrophil recruitment• Activation of Macrophages and Mast cells• Release of ILs, Chemokines

NK1, NK2

Indirect Actions • Angiogenesis• Stimulation of Adhesion molecules• Induces Apoptosis?


• Tissue homogenates in incubation buffer• Incubated with

• 3H-Substance P alone – Total Binding• 3H-Substance P + unlabelled Substance P (saturated) – Non-specific

Binding• 3H-Substance P + Test drug (subtract the non-specific binding)

• Two types – • Competition studies – increasing concentrations of Test drug• Saturation studies – increasing concentrations of 3H-Substance P

• Tissues – Chinese Hamster Ovary (CHO), CHO cells transfected with human ileum NK2 receptor, Guinea pig cortical membranes, human lymphoblasts,


Isolated Tissue Preparations for NK receptors

• Inhibition of Substance P-induced endothelium-dependent relaxation of rabbit pulmonary artery, previously contracted with 0.1 μM Noradrenaline

• Inhibition of Substance P or Substance P sulfone-induced contractions of guinea pig ileum in the presence of 3 μM Atropine and 3 μM Mepyramine and Indomethacin

• Inhibition of Substance P-induced plasma extravasation in guinea-pig bronchi


Polymorphonuclear Leukocyte Chemotaxis

• Measures the chemotactic effects on the PMN Leukocytes




Polymorphonuclear Leukocyte Chemotaxis

• Measures the chemotactic effects on the PMN Leukocytes

• Migration rate – number of PMNs in the lower well x 100number of PMNs in the upper well

• Dependent on the concentration of the chemoattractant (e.g., zymosan-activated serum)


PMN leukocytes aggregation induced by FMLP

• FMLP (formyl-L-methionyl-Lleucyl- L-phenylalanine)• PMNs cell suspensions + test compounds or standard

(pentoxiphylline) are dissolved in GBSS • Incubated for 10 min• Serial dilutions of FMLP added• Change in transmittance


Constitutive and inducible cellulararachidonic acid metabolism in vitro

• Formation of leukotriene B4 in human white blood cells

• Formation of lipoxygenase products from 14C-arachidonic acid in human polymorphonuclear neutrophils (PMN)

• Formation of eicosanoids from 14C-arachidonic acid in human platelets

• Stimulation of inducible prostaglandin pathway in human PMNL


COX-1 and COX-2 inhibition

• Inhibition studies with recombinant human COX-1 and COX-2

• HPLC assay for oxygenation of radiolabelled arachidonic acid by COX-1

• Determination of the stoichiometry of inhibitor binding

• Spectrophotometric assay of recombinant human COX-2

• Whole-cell assays with transfected Chinese hamster (CHO) cells expressing COX-1 and COX-2


Induced release of cytokines from human white blood cells

• Interleukin-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha• Cell fraction from whole blood separated • Exposed to LPS (Salmonella abortus equii) with or

without test drug• Incubation• Analysis of Cytokines


Flow cytometric analysis of intracellularcytokines

• Very powerful tool in which individual cells can be simultaneously analyzed for several parameters:• Size, granularity• Expression of surface and intracellular markers

• Defined by fluorescent antibodies. • Fluorescent anti-cytokine and anti-chemokine

monoclonal antibodies


In Vivo Methods


• Based on the symptoms observed during the inflammation• Acute/Transient phase: In this phase vasodilation

and increased capillary permeability are observed.• Subacute/Delayed phase : Infiltration of

leucocytes and phagocytes in blood.• Chronic inflammatory phase: granuloma formation

is observed in this phase


• Acute and Subacute phase:• Carrageenan induced paw oedema in rats• Croton-oil induced ear edema• Oxazolone induced ear edema • UV-B erythema in guinea pigs• Pleurisy in rats• Vascular permeability

• Chronic phase:• Cotton wool granuloma• Glass rod granuloma


Paw edema

• Most commonly employed method• Various substances used:• Carrageenin, Brewer’s yeast, Formaldehyde, Dextran, Egg

albumin, Kaolin, Zymosan• Serotonin, Histamine, Substance P, Cobra Venom

• Subplantar region Left Hind Paw (0.05 ml-SC)• Immediately after injection, again 3 and 6 h, and

eventually 24 h after challenge



• Biphasic Response:1st Phase – Hyperemia – Histamine, Serotonin, Kinins2nd Phase – Edema – Prostaglandins, Chemokines

• % Inhibition – Baseline volume – Volume at time x 100

Baseline volume


Ear edema

• Croton oil, Tetradecanoyl phorbol acetate, Xylol, Oxazolone

• Topical application• Whole ear, punched discs, caliper, other ear


Ultraviolet B erythema in guinea pigs

• Erythema – earliest sign of inflammation• Depilated skin exposed to UV-B rays for 20-30 secs.• Degree of erythema estimated after 2 hours

visually• 0 = no erythema• 1 = mild erythema• 2 = moderate erythema• 3 = strong erythema• 4 = very strong erythema



• Pure measure of the vasodilatory phase• No accompanying exudation or edema• Drawbacks:• Variable response• Accurate measurement of response

• Other sites/animals - • Guinea pig ear• Sprague Dawley rats• Hairless mice (skin thickness)


Pleurisy test

• Exudative Inflammation

• Histamine, Bradykinin, Prostaglandins, Dextran, Antigens, Microbes, irritants like turpentine and carrageenan etc.

• Fluid extravasation, leukocyte migration, lysosomal enzyme activities etc.


Vascular permeability

• Inhibitory activity of drugs against increased vascular permeability

• Extravasation of dye into surrounding tissue


Cotton wool granuloma

• Foreign body granuloma• Sterile Cotton pellets embedded

subcutaneously• Back, Axilla, Groin• After 7 days – Pellet + Granuloma removed• Weight of granulomatous tissue =Dry weight of granuloma cotton – initial weight

of cotton pellet



Glass Rod granuloma

• Foreign body granuloma• Glass peices embedded subcutaneously• Back, Axilla, Groin• After 20-40 days – Granuloma + Connective

removed entirely • Glass rod can be removed• Tensile strength and wet weight of

granulomatous tissue can be measured



Side Effects of Anti-inflammatory Agents


Ulcerogenic effect in rats

• Gastric irritation• 48 hrs fasting• 3, 5, 7 hrs after drug administration –

sacrificed• Check gastric lining for hyperemia and ulcers


Measurement of gastric mucosal damageby intragastric inulin

• Inulin in the gastric wall – measure of gastric damage

• Given intragastric• 3H-inulin radioactivity is expressed as

disintegrations/min/ml of plasma or per gram of tissue


Determination of blood loss

• Microbleeding which is not detected by observation of overt ulcers

• Feces, Stomach and intestinal contents

• Heme - spectrophotometry


Specific Disease Process


Adjuvant Arthritis in Rats

• Mimics human rheumatoid arthritis• Freund’s Adjuvant – Mycobacterium

butyricum• Delayed hypersensitivity response to

mycobacterial antigen• Subplantar region of left hind paw


• Day 1 Day 4 Day 7Local Peak Systemicreaction disease

• Parameters :• Paw edema, weight, grip strength , deformity

measured• Evaluation of chronically administered drug

against inflammation• On Day 5 and 21: Plethysmometer


• On Day 21:ears: absence of nodules and redness . . . . . . . . . 0

presence of nodules and redness . . . . . . . . 1nose: no swelling of connective tissue . . . . . . . . . 0

intensive swelling of connective tissue . . . 1tail: absence of nodules . . . . . . . . . . . . . . . . . . . . . 0

presence of nodules . . . . . . . . . . . . . . . . . . . . . 1forepaws: absence of inflammation . . . . . . . . . . . . 0

inflammation of at least 1 joint . . . . . . . 1hind paws: absence of inflammation . . . . . . . . . . . 0

slight inflammation . . . .. . . . . . . . . . . . . 1moderate inflammation . . . . . . . . . . . . . 2marked inflammation . . . . . . . . . . . . . . . 3


• Day 5 : Primary Lesions• Day 21 : Secondary Lesions

• Arthritic Index : Sum of scores of each animal

• Total % change = percent inhibition of the injected paw on day 5 + percent inhibition of the non-injected paw on day 21 + percent change of the arthritic index


Schultz-Dale Reaction

• Anti-anaphylactic activity• Guinea pigs are sensitized against egg

albumin. • Challenge after 3 weeks causes in isolated

organs release of mediators, e.g. histamine, which induce contraction in isolated ileum


Adhesion Assays

• VCAM-1,ICAM-1 and E-selectin• HUVEC ,HDMEC cell lines• Cell based ELISA – assay

• Endothelial Cell – Neutrophil adhesion• Platelet – Neutrophil adhesion


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Experimental evaluation of anti inflammatory agents